text: Biosynthesis of msDNA is purported to follow a unique pathway found nowhere else in DNA/RNA biochemistry. Because of the similarity of the 2-5 branch junction to the branch junctions found in RNA splicing intermediates, it might at first have been expected that branch formation would be via spliceosome- or ribozyme-mediated ligation. Surprisingly, however, experiments in cell-free systems using purified retron reverse transcriptase indicate that cDNA synthesis is directly primed from the 2-OH group of the specific internal G residue of the primer RNA. The RT recognizes specific stem-loop structures in the precursor RNA, rendering synthesis of msDNA by the RT highly specific to its own retron. The priming of msDNA synthesis offers a fascinating challenge to our understanding of DNA synthesis. DNA polymerases (which include RT) share highly conserved structural features, which means that their active catalytic sites vary little from species to species, or even between DNA polymerases using DNA as a template, versus DNA polymerases using RNA as a template. The catalytic region of eukaryotic reverse transcriptase comprises three domains termed the "fingers", "palm", and "thumb" which hold the double-stranded primer-template in a right-hand grip with the 3-OH of the primer buried in the active site of the polymerase, a cluster of highly conserved acidic and polar residues situated on the palm between what would be the index and middle fingers. In eukaryotic RTs, the RNase H domain lies on the wrist below the base of the thumb, but retron RTs lack RNase H activity. The nucleic acid binding cleft, extending from the polymerase active site to the RNase H active site, is about 60 Å in length in eukaryotic RTs, corresponding to nearly two helical turns. When eukaryotic RT extends a conventional primer, the growing DNA/RNA double helix spirals along the cleft, and as the double helix passes the RNase H domain, the template RNA is digested to release the nascent strand of cDNA. In the case of msDNA primer extension, however, a long strand of RNA remains attached to the 3-OH of the priming G. Although it is possible to model an RT-primer template complex which would make the 2-OH accessible for the priming reaction, further extension of the DNA strand presents a problem: as DNA synthesis progresses, the bulky RNA strand extending from the 3'-OH needs somehow to spiral down the binding cleft without being blocked by steric hindrance. To overcome this issue, the msDNA reverse transcriptase clearly would require special features not shared by other RTs.